Development of New Proteomics Technology and its Application to Study Cellular Organization The broad goal of our lab is to obtain a systems level understanding of cellular organization and develop proteomics technology that facilitates this research. Thanks to the human genome project, we have a near complete parts list of all molecules making up cells, but we still very poorly understand how all these molecules come together and organize marvelously into a living system. So far this organization has been mostly studied by looking carefully at one protein at a time. While this approach has been tremendously successful, it cannot address the higher levels of complexity in biological systems that arises from the interplay of a myriad of components. Looking at one molecule at a time seems to be serious hindrance towards understanding biology. Rather, we have to start investigating the entire system all at once. Recent progress in multiplexed proteomics enables us to observe thousands of proteins simultaneously among multiple conditions. In combination with classical biochemical approaches this allows us to reveal collective behavior and emergent properties we would have no chance to discover otherwise. My lab is broadly interested in systems-level cellular organization. Towards this goal, this proposal contains two parts. The first part describes how we aim to decipher how the proteome partitions between nucleus and cytoplasm in early development and decode how the embryos uses differential nuclear composition for the appropriate timing of transcription and nuclear size control. The second part of this proposal outlines how we intend to improve quantitative proteomics technology. Proteomics has become very powerful. Nevertheless, severe shortcomings in respect to sensitivity, data quality and accessibility remain. We strive to address these problems. Over the last year, we have developed a new method for quantitative shotgun proteomics (TMTc+), which produces data with unmatched sensitivity and measurement quality while reducing cost. Next, we aim to combine TMTc+ with a targeted approach. We anticipate that this will enable us to quantify proteins down to the lowest abundant transcription factors. This new technology will benefit our own research of cellular organization, but more generally will benefit researchers that are interested in differential protein expression levels to study basic biology or disease.